Abstract

Blepharophimosis syndrome (BPES), an autosomal dominant syndrome in which an eyelid malformation is associated (type I) or not (type II) with premature ovarian failure (POF), has recently been ascribed to mutations in FOXL2, a putative forkhead transcription factor gene. We previously reported 22 FOXL2 mutations and suggested a preliminary genotype-phenotype correlation. Here, we describe 21 new FOXL2 mutations (16 novel ones) through sequencing of open reading frame, 5' untranslated region, putative core promoter, and fluorescence in situ hybridization analysis. Our study shows the existence of two mutational hotspots: 30% of FOXL2 mutations lead to polyalanine (poly-Ala) expansions, and 13% are a novel out-of-frame duplication. In addition, this is the first study to demonstrate intra- and interfamilial phenotypic variability (both BPES types caused by the same mutation). Furthermore, the present study allows a revision of the current genotype-phenotype correlation, since we found exceptions to it. We assume that for predicted proteins with a truncation before the poly-Ala tract, the risk for development of POF is high. For mutations leading to a truncated or extended protein containing an intact forkhead and poly-Ala tract, no predictions are possible, since some of these mutations lead to both types of BPES, even within the same family. Poly-Ala expansions may lead to BPES type II. For missense mutations, no correlations can be made yet. Microdeletions are associated with mental retardation. We conclude that molecular testing may be carefully used as a predictor for POF risk in a limited number of mutations.

The predicted protein translations for FOXL2 mutations detected in this and previous studies are shown at the left and are assembled into groups. Groups A–D are predicted truncated proteins: A, without forkhead domain; B, with partial forkhead; C, with complete forkhead and without poly-Ala tract; D, with complete forkhead and poly-Ala domains. Group E comprises frameshift mutations leading to elongated proteins with complete forkhead and poly-Ala domains; group F, in-frame mutations; and group G, missense mutations. Vertical stripes indicate the forkhead domain, dark gray shows the poly-Ala tract, and diagonal stripes represent novel amino acids caused by a frameshift mutation. In the first column, the corresponding nucleotide changes are represented. The numbering is according to Crisponi et al. (). Mutations found in the present study are indicated in bold, and novel mutations are underlined. In the second column, the respective amino acid changes are shown. The two mutational hotspots demonstrated in the present study are boxed, and their percentages are shown at the right: 30% of the mutations lead to poly-Ala expansions, and 13% are a novel duplication 1080–1096dup17. Abbreviations: fs = frameshift; stop = stop codon. In the third column, the BPES type is shown (F1 = familial, type I; F2 = familial, type II; F1+2 = familial, occurrence of both BPES types in the same family; F = familial, type undetermined; S1 = sporadic, type I; and S = sporadic).

Explanation of the mechanism of the novel triplication 921–935trip15 by a replication error. Predictions of DNA hairpins are based on (Walter et al. ). When the DNA polymerase is stalling because of the GC richness of the region, the sequences 1 and 2 of the newly synthesized strand form a DNA hairpin because they are self-complementary (structure 1). This allows the sequence 3 to hybridize with the template of the region 2, since they have exactly the same sequence, and this leads to a duplication of the underlined sequence (nt 921–935). Once this extra sequence is present (4), there may be a rearrangement of the hairpin, which leads to the formation of another one involving the sequences (single and double asterisk). The formation of the second hairpin is favorable, because it is more stable (by an energy excess of 1.5 kcal/mole; see structure 2). Finally, the 3′ end of the sequence 4 hybridizes with the template of sequence 2, which results in the triplication after escaping repair. Further, notice that sequences 1–4 form a very stable, almost perfect, hairpin (−28 kcal/mole).